Three-dimensional (3D) laser scanning technologies have great importance in analysing biological specimens e.g. imaging 3D biological structures or mapping fluorescent markers of cell surface receptors on non-planar surfaces.
Commonly used 3D laser scanning microscopes are either confocal microscopes or two-photon (or multi-photon) microscopes. In the confocal microscope technology a pinhole is arranged before the detector to filter out light reflected from any other plane than the focus plane of the microscope objective. Thereby it is possible to image planes lying in different depths within a sample (e.g. a biological specimen).
Two-photon laser scanning microscopes use a laser light of lower energy of which two photons are needed to excite a flourophore in a quantum event, resulting in the emission of a fluorescence photon, which is then detected by a detector. The probability of a near simultaneous absorption of two photons is extremely low requiring a high flux of excitation photons, thus two-photon excitation practically only occurs in the focal spot of the laser beam, i.e. a small ellipsoidal volume having typically a size of approximately 300 nm×300 nm×1000 nm. Generally a femtosecond pulsed laser is used to provide the required photon flux for the two-photon excitation, while keeping the average laser beam intensity sufficiently low.
In order to decrease the required scanning time the laser beam is preferably deflected by known means to scan different points of a given focal plane (XY plane). Several known technologies exist for deflecting the laser beam prior to entering the objective, e.g. via deflecting mirrors mounted on galvanometric scanners, or via acousto-optical deflectors.
The galvanometric scanners and the acousto-optical deflectors are very fast devices, hence moving the focus spot to a desired XY plane position and obtaining measurement data via the detector in that position can be carried out in less than 1 ms.
Even two-dimensional (2D) scanning technologies commonly used for scanning and/or imaging thin specimen slides usually involve focal plane changing as well, i.e. the suitable slide-objective distance needs to be found for obtaining a sharp image since the optimal focus position may vary from slide to slide and even within the same specimen slide.
Focal plane changing (3D sampling) can be carried out by moving the sample stage and the focusing means (typically the objective) of the microscope relative to each other. This is either realised by moving the sample stage e.g. via stepping motors, or by displacing the microscope objective. Displacing the objective is generally regarded as being disadvantageous since this involves moving a greater mass, which implies setting the greater mass in motion and stopping it at the desired position. Accordingly, it is preferred to move the sample stage instead, which allows for much faster Z-scanning (scanning along the optical axis).
However, moving the stage is complicated to implement when using submerge specimen chambers or when electrical recording is performed on the biological specimen with microelectrodes. Accordingly, in the case of analysing biological specimens it is often preferred to move the focus spot of the laser beam instead of moving the specimen. This can be achieved by deflecting the laser beam to scan different points of a focal plane (XY plane) and for example by displacing the objective along its optical axis (Z axis) e.g. via a piezo-positioner to change the depth of the focal plane.
Information about the sample is provided by detecting the scanning beam reflected back from the specimen or in case of fluorescent microscopy by detecting the fluorescence light. Suitable detectors and measuring methods and corresponding image generating devices (e.g. detector, computer and monitor) are well known in the art. As explained above prior art efforts were directed to reduce the mass that is to be displaced during focusing. Accordingly, the detectors, which represent a considerable mass, were held in a fixed position while the objective was displaced relative to the sample and to the detector, or the sample was displaced relative to the objective and detector.
A typical prior art laser scanning microscope construction is illustrated in FIG. 1. As can be seen the laser scanning microscope 100 comprises a laser source 112 providing a laser beam 113 which is focused onto a sample 122 (e.g. a biological specimen) via microscope objective 116. In order to perform 3D sampling through focal plane change a drive 118 is provided for displacing the microscope objective 116 relative to the sample 122. A beam splitter 123 is arranged along the optical path of the scanning laser beam 113 for directing the reflected light 113′ to a suitable detector 124. In order to move as little mass as possible the detector 124 is in a fixed position and only the objective 116 is displaced by the drive 118.
The first major drawback of this solution is that the optical distance between the objective 116 and the detector 124 needs to be relatively big, typically a 30-40 mm gap 101 is required between the top of the objective 116 (or the system supporting the objective 116) and the beam splitter 123 in order to be able to lift the objective when arranging the sample 122 on the stage. Such a long optical distance is particularly undesirable in the case of fluorescence microscopy where the scattered nature of the back fluoresced light 113′ can lead to high losses along the relatively long optical path.
A second drawback of the prior art solution is associated with the varying optical distance between the objective 116 and the detector 124 when measuring biological specimens arranged at different height and/or when performing focal depth changing. This may lead to a fluctuation in the detected light intensity because varying optical distance means varying light intensity loss along the optical path even where the reflected light 113′ is a reflected laser beam. Furthermore, detection and/or imaging of the specimen is rendered more complicated if the distance between the objective 116 and the detector 124 is not constant.
U.S. Pat. No. 5,132,526 discloses a microscope, which is based on the general prior art principle, i.e. only the objective is displaced relative to the sample, while the detectors are held in a fixed position. The problem of relatively long and varying optical distance between the detectors and the objective is overcome by providing the optical path partly inside an optical fiber, whereby a constant optical distance is maintained between the detectors and the objective. However, the use of an optical fiber only allows for detecting an image of the focus spot, meaning a confocal detection arrangement, since the same optical fiber is used to direct exciting light on the sample. In a confocal type detection the sample side focal plane of the objective is imaged onto the aperture placed immediately in front of the detector, hence only light propagating parallel to the objective axis will be detected. However, in case when all scattered reflected and fluorescent light contains useful information about the sample—e.g. in the case of two-photon microscopy—all light transmitted by the objective from the sample should be captured by the detector for best performance.
Due to the use of the optical fiber only a smaller fraction of all the light originating from the focus spot can be detected, hence this arrangement is not suitable for carrying out a non-confocal detection method as necessary e.g. in two-photon microscopy. It should be noted that although U.S. Pat. No. 5,132,526 also discloses a 4-segment photodetector that is displaced together with the objective, this photodetector is used to adjust focusing in a confocal arrangement. In order to achieve this task the pinhole from the front of the photodetector is removed and the detector is arranged to detect an image of the focus spot of the illuminating light in the sample, which allows for detecting whether or not the light is focused on a specific plane in the specimen.
It is an object of the present invention to overcome the problems associated with the prior art laser scanning microscopes and to provide a laser scanning microscope wherein the objective-detector distance is minimised and can be kept substantially constant regardless of the shape and height of a specimen to be examined, and optionally in the course of information collection from a 3D volume, while increasing the intensity of the detected light by detecting scattered, reflected or fluorescence light as well.
The present invention is based on the recognition that the above problems can be overcome by mounting an objective and an image detector arranged to detect an image from the back aperture of the objective on a common drive that simultaneously displaces both the objective and the detector. The objective and detector assembly are referred to as a “focusing-detecting unit”.
It is a further object of the present invention to provide a 3D laser scanning microscope with the above advantages.
In “Imaging cellular network dynamics in three dimensions using fast 3D laser scanning” (Nature Methods, Vol. 4 No. 1, January 2007) Göbel et al. propose to drive a piezo-positioner of a laser scanning microscope objective with a sinusoidal signal and calculate an appropriate drive signal for the X-Y scanners (galvanometric scan mirrors) to obtain a desired 3D trajectory. The article discusses measurements made at a sinusoidal drive signal of 10 Hz and suggests adjustment of the drive signal to compensate for amplitude reduction and phase shift of the actual objective position with respect to the drive signal of the piezo-positioner.
One of the problems associated with the above method is a deviation from the desired scan trajectory because the movement of the objective deviates from sinusoidal owing to the properties of the piezo-positioner and other mechanical components. This problem is not crucial at low frequencies of the sinusoidal drive signal, such as the 10 Hz frequency used by Göbel et al. However, the deviation becomes more and more significant as the frequency is increased.
Being constrained to use low frequencies is less disturbing when scanning a large number of X-Y positions in each scanning plane (i.e. planes lying at different Z depths within the specimen) as the fast XY positioning allows for obtaining a plurality of scans while the focus plane remains substantially in the same Z plane. Thus, effectively, the relatively long time spent in each scanning plane is not wasted as a plurality of measurements can be carried out. On the other hand, when scanning specimens having only a few points of interest in each Z plane, e.g. a nerve cell dendrite crossing such planes, the aim is to spend as little time in each Z plane as possible in order to decrease the overall scan time. Therefore it would be desirable to increase the frequency of the sinusoidal drive signal but as indicated by Göbel et al., such an increase in the frequency would result in a higher deviation between the displacement of the objective and a theoretical sinusoidal displacement corresponding to the sinusoidal drive signal, which could lead to an intolerable deviation from the desired 3D scanning trajectory, effectively the positions of interest within the specimen could be out of focus or could be missed entirely.
It is a second object of the present invention to overcome the above problem and provide a method for decreasing the Z-scanning time when performing 3D sampling by moving the whole of the focusing-detecting unit in accordance with the inventive laser scanning microscope construction. It is a further object to provide a scanning method capable of compensating for a deviation between the motion of the focusing-detecting unit connected to a drive and the drive signal of the drive.